title: SnRK2.6/OST1 from Arabidopsis thaliana: cloning, expression, purification, crystallization and preliminary X-ray analysis of K50N and D160A mutants.
abstract: The SnRK2.6 (SNF1-related kinase 2.6) gene from Arabidopsis thaliana encodes the serine/threonine protein kinase SnRK2.6/OST1 (OPEN STOMATA 1). It plays a central role in the drought-tolerance mechanism. OST1 is in fact the main positive effector in the hydric stress response. The SnRK2.6 gene was cloned into the pGEX4T1 plasmid, mutated and expressed in Escherichia coli, allowing purification to homogeneity in two chromatographic steps. Various OST1 mutants yielded crystals using vapour-diffusion techniques, but only one mutant showed a good diffraction pattern. Its crystals diffracted to 2.8 Å resolution and belonged to space group P222(1), with unit-cell parameters a=77.7, b=99.4, c=108.4 Å. A promising molecular-replacement solution was found using the structure of the kinase domain of the yeast AMP-activated protein kinase SNF1 (PDB entry 3hyh) as the search model.
authors.affiliation: Departamento de Cristalografía y Biología Estructural, Instituto de Química Física Rocasolano, Consejo Superior de Investigaciones Científicas, Serrano 119, 28006 Madrid, Spain. xyunta@iqfr.csic.es
authors.forename: Cristina
authors.initials: C
authors.lastname: Yunta
authors.forename: Martin
authors.initials: M
authors.lastname: Martinez-Ripoll
authors.forename: Armando
authors.initials: A
authors.lastname: Albert
mesh_terms: D017360:Arabidopsis / Q000737:chemistry*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification*; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification*; D014961:X-Ray Diffraction
chemical_list: D029681:Arabidopsis Proteins; D011494:Protein Kinases; C472139:OST1 protein, Arabidopsis